It is recognized that individual humans differ in their sequence and recently several individuals have had their genomes sequenced, for instance James Watson and Craig Venter. Comparison of the genome sequence of individuals has revealed differences in their sequences in both coding and non-coding parts of the genome. Some of these variations in humans are significant and contribute to phenotypic differences between individuals. In extreme cases these will result in genetic disease. The 1000 Genomes Project has the objective of cataloguing sequences in the human genome, involving sequencing the genomes of a very large sampling of individuals from diverse art-recognized human ethnic populations.
Interleukin-6 (IL-6) is a complex cytokine, which plays a critical role in the regulation of inflammatory responses. Interleukin-6 (IL-6) signals through a ligand-binding IL-6 receptor (IL-6R; also known as CD126) chain and a common signal-transducing chain glycoprotein 130 (gp130; also known as CD130), which is also engaged by receptors specific for IL-11, IL-27, leukaemia inhibitory factor, oncostatin M (OSM), ciliary neurotrophic factor (CNTF) and cardiotrophin 1 (CT1). A soluble form of the IL-6R (sIL-6R) can be generated by proteolytic cleavage of mIL-6R by the metalloproteinases TNFα-converting enzyme (TACE; also known as ADAM17) and ADAM10 or alternatively spliced mRNA. IL-6 responses can be induced classically in cells expressing mIL-6R through a high-affinity tetrameric complex consisting of IL-6, IL-6R and two gp130 molecules (or a hexameric complex consisting of two IL-6, two mIL-6R and two gp130 molecules). Alternatively, a sIL-6-IL-6R complex directly binds to and signals through gp130 in cells lacking mIL-6R in a process that has been termed trans-signalling. High levels of IL-6 and sIL-6R have been reported in several chronic inflammatory diseases and in cancer. Several drugs that target different components of the IL-6 and IL-6R system have been described, including IL-6-specific monoclonal antibodies (mAbs) (for example, CNTO 328), IL-6R-specific mAbs (for example, tocilizumab) and soluble gp130-Fc, an antagonist of IL-6R trans-signalling. See a review at “Averting inflammation by targeting the cytokine environment”, Manfred Kopf, Martin F. Bachmann & Benjamin J. Marsland, Nature Reviews Drug Discovery 9, 703-718 (September 2010), doi:10.1038/nrd2805 (incorporated herein by reference).
Genetic variation in the IL-6 receptor gene is associated with the risk of several human diseases with an inflammatory component, including coronary heart disease, rheumatoid arthritis, and asthma. A non-synonymous variant in the IL-6 receptor gene (IL6R Asp358Ala; rs2228145 A>C) is associated with the increased susceptibility to asthma. It is thought that the variant exerts its functional mechanism by regulating the balance between sIL6R (generated through cleavage of the cell surface receptor and by alternative splicing of a soluble IL6R isoform) and membrane-bound IL-6R (involved in classic IL6R signalling). Ferreira et al reportedly showed for the first time that the minor allele of this non-synonymous variant (Ala358) directly controls the surface levels of IL-6R on individual immune cells and that these differences in protein levels translate into a functional impairment in IL6R signaling.
Ferreira et al noted that while classic IL6R signalling appears responsible for regulatory T-cell suppression, IL6R trans-signalling (via sIL6R) seems to promote T helper 2 cell polarization in the lung. IL6R has been shown to be expressed in the epithelium, smooth muscle and vascular endothelium of human airways, and in macrophages and granulocytes of bronchioalveolar lavage fluid (BALF). Soluble IL6R levels in BALF are elevated in asthmatic patients compared to controls, and are elevated further upon allergen challenge, indicating an important role of sIL6R in the context of the lung and associated tissues. Consistent with these findings, as noted above 358Ala is also associated with an increased severity of asthma. The authors also provide evidence for the association of this non-synonymous variant with the risk of type 1 diabetes (T1D) in two independent populations and confirm that rs2228145 is the major determinant of the concentration of circulating soluble IL6R (sIL6R) levels (reportedly 34.6% increase in sIL6R per copy of the minor allele 358Ala; rs2228145 [C]). The authors provided evidence that the non-synonymous variant rs2228145 regulates the balance of surface and sIL6R, and also affects the responsiveness of immune cells to IL6 stimulation. This mechanism underpins the effect of 358Ala on the IL-6/IL-6R pathway.
Revez et al comments that the main genetic determinant of soluble interleukin 6 receptor levels is the variant rs2228145 that maps to the cleavage site of IL6R. Reportedly, for each Ala allele, sIL6R serum levels increase by about 20 ng ml and asthma risk by 1.09-fold. However, the authors conclude that this variant does not explain the total heritability for sIL6R levels. Additional independent variants in IL6R may therefore contribute to variation in sIL6R levels and influence asthma risk. The authors imputed 471 variants in IL6R and tested these for association with sIL6R serum levels in 360 individuals. An intronic variant (rs12083537) was associated with sIL6R levels independently of rs4129267 (P=0.0005), a proxy single-nucleotide polymorphism for rs2228145. A significant and consistent association for rs12083537 was observed in a replication panel of 354 individuals (P=0.033). Each rs12083537:A allele increased sIL6R serum levels by 2.4 ng ml. Analysis of mRNA levels in two cohorts did not identify significant associations between rs12083537 and IL6R transcription levels. On the other hand, results from 16705 asthmatics and 30809 controls showed that the rs12083537:A allele increased asthma risk by 1.04-fold (P=0.0419).
J C Galicia et al investigated the association of the IL6R polymorphisms with the serum levels of soluble IL6 receptor. In total, 115 healthy volunteers were genotyped, with 70 of them analyzed for sIL6R levels. Using the PCR/RFLP methods, two important polymorphic sites were selected for genotyping: the 48892A/C (D358A) in exon 9 and the −183G/A in the promoter region. In exon 9, C allele carriers had higher sIL6R level (P<0.0001) showing that this sequence variation, which corresponds to the proteolytic cleavage site of IL-6Ralpha, strongly influences the serum sIL6R levels. In the promoter region, G allele carriers had lower sIL6R levels (P<0.0082) compared with the A allele carriers.
Esparza-Gordillo et al examined the results of all genome-wide association studies from a public repository and selected 318 genetic markers that were significantly associated with any inflammatory trait. These markers were considered candidates and tested for association with atopic dermatitis (AD) in a 3-step approach including 7 study populations with 7130 patients with AD and 9253 control subjects. Reportedly a functional amino acid change in the IL-6 receptor (IL-6R Asp358Ala; rs2228145) was significantly associated with AD. Investigation of 2 independent population-based birth cohorts showed that IL-6R 358Ala specifically predisposes to the persistent form of AD. Carriers of 358Ala had increased serum levels of soluble IL6R, with homozygote carriers showing a 2-fold increase. Moreover, the authors reported that sIL6R levels were higher in patients with AD than in control subjects. The authors conclude that the study supports the importance of genetic variants influencing inflammation in the aetiology of AD. Moreover, they identified a functional genetic variant in IL6R influencing disease prognosis and specifically predisposing to persistent AD.
Antibodies to human IL6R are described in U.S. Pat. No. 8,043,617, U.S. Pat. No. 5,670,373, U.S. Pat. No. 5,817,790, EP409607 and those publications in Table 13 below. Therapeutic methods are described in U.S. Pat. No. 5,888,510, U.S. Pat. No. 8,043,617, U.S. Pat. No. 6,723,319 and those publications in Table 13 below. Actemra™ (tocilizumab) is a humanised example of an anti-IL-6R agent that blocks both classic and trans-signaling. When directed to antibodies, the invention instead is directed to antibodies with fully human (not humanised) variable regions (and optionally also human constant regions).
Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of synovial tissue, leading to destruction of the joint architecture. It is recognized that cytokines such as tumor necrosis factor (TNF), interleukin-1 (IL-1) and interleukin-6 (IL-6) play a role in joint inflammation and cartilage damage observed in RA. IL-6 is a pleiotropic cytokine with biological effects on many cell types. IL-6 is often regarded as being downstream of TNF or IL-1 in inflammatory cytokine cascades and may therefore represent a common pathway factor in a wide range of inflammatory processes. Blockade of IL-6 signaling therefore offers the potential to ameliorate multiple pathogenic features of RA and other inflammatory diseases.
Therapeutic methods using IL-6R antagonists are mentioned in U.S. Pat. Nos. 5,888,510; 6,723,319; and 2001/0001663. Exemplary anti-IL-6R antibodies are described in U.S. Pat. Nos. 7,582,298; 6,410,691; 5,817,790; 5,795,695; 6,670,373; and 7,582,298.
In certain embodiments, an IL6R polypeptide includes terminal residues, such as, but not limited to, leader sequence residues, targeting residues, amino terminal methionine residues, lysine residues, tag residues and/or fusion protein residues. “IL6R” has also been referred to as interleukin-6 receptor subunit alpha, CD126; IL-6R-1; IL-6RA; IL6Q; IL6RA; IL6RQ; and gp80.